Peroxidase is an enzyme that catalyzes the oxidation of various compounds such as phenols and amines, by peroxides. In addition, particular compounds have been termed pseudoperoxides because they behave in a manner similar to the peroxidase enzyme by liberating oxygen from hydroperoxides and transferring the oxygen to certain acceptor compounds. Accordingly, the pseudoperoxides are enzyme-like in that they catalyze, or otherwise participate in, reactions between peroxides and oxidizable compounds. The pseudoperoxides, which include hemoglobin and its derivatives, are regarded as peroxidatively active substances.
For example, in the assay of urine for glucose, the enzyme glucose oxidase, in the presence of oxygen, first converts the glucose in the urine to gluconic acid and hydrogen peroxide after which the peroxidase enzyme which is included in the assay system catalyzes the interaction between the hydrogen peroxide (hydroperoxide) and an oxidizable dye compound, such as o-tolidine or tetramethylbenzidine, to cause the dye, which is colorless in its reduced state, to become colored thereby providing a detectable response. The degree and intensity of the colored response are directly proportional to the amount of hydrogen peroxide generated by the glucose conversion, provided there is sufficient peroxidase present to catalyze the oxidation of the dye.
Similarly, a peroxidatively active substance such as hemoglobin or a derivative thereof can catalyze the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase and catalyzes the interaction between the oxidizable dye and the hydroperoxide. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity of the response is indicative of the concentration of the peroxidatively active substance.
Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency.
The first practical test for the determination of creatinine in urine, known as the Jaffe method, involves the formation of the red-yellowish brown colored creatinine picrate by the bonding of picric acid and creatinine in an alkaline solution. A more recent method for creatinine determination is reported by Benedict and Behre in J. Biol. Chem., 113: 515 (1936) which involves the reaction of 3,5-dinitrobenzoic acid with creatinine in an alkaline medium. Each of these reactions require a high pH, i.e. on the order of 12-13, in order to deprotonate the creatinine, so that the system can operate properly. Strongly basic substances such as alkali and alkaline earth metal hydroxides are typically used to maintain a suitably high pH in these reagent systems. Operating at such a high pH presents various difficulties, especially when an absorbent carrier such as filter paper or a porous film is used as carrier for the reagent system because, upon introduction of the alkali, the carrier tends to become brittle and it is difficult to obtain even distribution of the alkali throughout the carrier matrix. Furthermore, when the reagents are applied to the carrier in the form of a solution and the solvent evaporated to leave a dry residue, the dried alkali does not readily solubilize when contacted with a fluid such as urine which is being examined for creatinine concentration.
The present invention is predicated on the discovery that copper ion and creatinine are able to form a complex which will act as a pseudoperoxidase in the previously described system in which a hydroperoxide in the presence of a peroxidase or pseudoperoxidase oxidizes a chromogenic indicator to provide a detectable response.
In Polyhedron, Vol. 4, No. 7, Pp. 1159-1161, 1985; Mitewa et al describe a complex formation between Cu(II) and creatinine. They disclose that a Cu(II) complex with creatinine is formed with a metal:ligand ratio of 1:2 and that complex 4-membered chelate rings are formed which differ significantly from the corresponding Co(II), Cd(II), Zn(II) and Hg(II) complexes.